Sedimentation field flow fractionation is a versatile technique for the high resolution separation of a wide variety of particulates suspended in a fluid medium. The particulates including macromolecules in the 10.sup.5 to the 10.sup.13 molecular weight (0.0001 to 1 .mu.m) range, colloids, particles, unicelles, organelles and the like. The technique is more clearly described and more explicitly described in U.S. Pat. No. 3,449,938, issued June 17, 1969 to John C. Giddings and U.S. Pat. No. 3,523,610, issued Aug. 11, 1970 to Edward M. Purcell and Howard C. Berg.
Field flow fractionation is the result of the differential migration rate of components in a carrier or mobile phase in a manner similar to that experienced in chromatography. However, in field flow fractionation there is no separate stationary phase as is in the case of chromatography. Sample retention is caused by the redistribution of sample components between the fast to the slow moving strata within the mobile phase. Thus, particulates elute more slowly than the solvent front. Typically a field flow fractionation channel consists of two closely spaced parallel surfaces. A mobile phase is caused to flow continuously through the gap between the surfaces. Because of the narrowness of this gap or channel (typically 0.025 centimeters (cm)), the mobile phase flow is laminar with a characteristic parabolic velocity profile. The flow velocity is the highest at the middle of the channel and the lowest near the two channel surfaces.
An external force field of some type (the force fields include gravitational, thermal, electrical, fluid cross flow and others described variously by Giddings and Berg and Purcell), is applied transversely (perpendicular) to the channel surfaces or walls. This force field pushes the sample components in the direction of the slower moving liquid strata near the outer wall. The buildup of sample concentration near the wall, however, is resisted by the normal diffusion of the particulates in a direction opposite to the force field. This results in a dynamic layer of component particles, each component with an exponential-concentration profile. The extent of retention is determined by the time-average position of the particulates within the concentration profile which is a function of the balance between the applied field strength and the opposing tendency of particles to diffuse.
In sedimentation field flow fractionation, use is made of a centrifuge to establish the force field required for the separation. For this purpose, a long, thin annular belt-like channel is made to rotate within a centrifuge. The resultant centrifugal force causes components of higher density than the mobile phase to settle toward the outer wall of the channel. For equal particle density, because of their higher diffusion rate, smaller particulates will accumulate into a thicker layer against the outer wall than will larger particulates. On the average, therefore, larger particulates are forced closer to the outer wall.
If now the fluid medium, which may be termed a mobile phase or solvent is fed continuously in one end of the channel, it carries the sample components through the channel for later detection at the outlet of the channel. Because of the shape of the laminar velocity profile within the channel and the placement of particulates in that profile, solvent flow causes smaller particulates to elute first, followed by a continuous elution of sample components in the order of ascending particulate mass.
One of the problems encountered in the use of thin channels is that the finish of the channel walls must be exceedingly smooth or else the separated particles, while undergoing their Brownian motion and under the influence of centrifugal force, can contact the wall and sometimes become stuck in the various interstices of such walls. At the very least, this tends to impede the flow of the particulates through the channel as desired and hence reduces the efficiency of the separation. One can tend to overcome a large part of this problem by making an extremely smooth wall finish. However, this is expensive and not always possible.
A related problem is that it is extremely difficult to make the relatively thin channels of constant radius so that they are perfectly circular. If the channel is not circular, the points of the channel that lie in a greater radial distance will be subjected to a different centrifugal force than those of a lesser radial distance. This leads to further decreases in the efficiency and resolution of the separation.
A final but related problem, which exists when multiple channels are used in side by side relation, is the leakage that can occur between the channels. This also leads to a degradation of result.